Bone Marrow is the most frequent site of the systemic spread of some types of cancer, including breast1,2 neuroblastoma, colorectal, and prostate cancer. Once metastases are clinically apparent, overall prognosis is poor. Undetected occult tumor cells contribute to disease recurrence and therefore methods to identify subclinical disease (micrometastasis) may improve staging and guide additional therapeutic decisions. Historically, conventional cytologic assessment of blood and bone marrow (BM) aspirates has been performed with limited success3,4. Immunocytochemical techniques using antibodies specific to epithelial antigens have improved sensitivity and can identifying a single tumor cell amongst a background of >1 million normal cells5,6. Enrichment methods with antibody-magnetic bead conjugates of BM aspirates have demonstrated the presence of occult tumor cells in early stage breast cancer patients5,7.
Recently it has been shown that the detection of micrometastasis in the BM of early stage breast cancer patients is an independent prognostic risk factor8,9. However, the accurate microscopic analysis of many cytologic samples requires considerable cytopathologic expertise and can be tedious, particularly if performed serially to assess disease progression and/or response to treatment. Additionally, the variable specificity of individual antibodies used to detect single cells has been questioned8,10,11. Finally, these assay systems cannot characterize the biologic behavior of the cells being detected and thus many may represent dormant tumor cells, apoptotic cells, nonpathologic tumor cells, or displaced normal breast epithelial cells.
A variety of serial genetic changes have been implicated in the initiation and progression of solid tumors. One such event, allelic imbalance (loss of heterozygosity; LOH) has been shown to occur commonly in primary breast tumors and with additional frequency in metastasis12-15. Furthermore, there is emerging evidence to suggest that microsatellite markers for detecting LOH at specific chromosome loci may have important clinical prognostic correlations12,16,17. However, the examination of an excised primary tumor specimen may be of limited value in that it provides information of those genetic events that have occurred and not ongoing alterations which may be of clinical relevance, either prognostically or for therapeutic decisions. Additionally, because of the potentially long latent period that may exist between early breast cancer diagnosis and clinically detectable systemic recurrence, improved assessment methods are needed for serial surveillance of occult disease progression and monitoring response to therapy.
Recently it has been shown that free tumor-associated DNA can be identified in the serum and plasma from patients with melanoma, breast, lung, renal, gastrointestinal, and head and neck tumors18-32. Furthermore a high-quality concordance has been shown to exist between the genetic alterations (i.e., LOH, microsatellite instability, mutations) found in circulating tumor DNA and those from the primary tumor suggesting a potential surrogate tumor marker20-22,27,28. Early studies have shown the prognostic importance of circulating microsatellite markers for LOH in blood21,24. Although, BM is a common site for recurrence of some types of cancer, such as breast and prostate cancer, to date, BM has not been studied for the presence of suitable genetic markers.
Recently, methylation of gene promoter regions and the role that this epigenetic event plays in the development of various cancers has become an important area of investigation in assessing the mechanisms of tumor suppressor and regulatory gene inactivation. The tumor suppressor genes (TSG) can be transcriptionally silenced when their promoter region CpG islands contain methylated cytosines located 5′ to an adjacent guanine. The utilization of methylation-specific PCR (MSP) assay has simplified and significantly improved detecting hypermethylated CpG bases with minimum amount of DNA. The methylation status of several TSG promoter regions have been profiled for a number of cancers. The hypermethylation of CpG islands of promoter regions of TSG is quite common and is a significant genetic aberration for tumor cells to shut off TSG expression.
Majority of these studies have been focused on carcinomas; there are limited studies in cutaneous melanomas and other types of cancers. The studies of epigenetic inactivation of TSG in melanomas have been limited mostly to methylation of promoter regions of p16INK4a and MGMT (O6-methylguanine-DNA methyltransferase). Interestingly, the frequency of TSG inactivation or mutations in oncogenes is reportedly low in cutaneous melanomas. These observations have been a major enigma in deciphering the genetic events occurring in melanoma progression.